This invention relates to imaging of cells and of gene expression.
A number of different approaches to imaging cells and gene expression have been investigated using either optical imaging techniques, e.g., using green fluorescent protein, bioluminescence, or near infrared fluorescence, or nuclear imaging techniques. However, these techniques may have limited depth penetration (optical techniques) or spatial resolution (nuclear techniques). Magnetic resonance (MR) imaging has also been used, and recent advances in MRI, and in particular MR microscopy, have led to improved image resolution. However, compared to current optical and nuclear techniques, molecular probe detection by MR is several magnitudes less sensitive.
The invention is based on the discovery that cells can be imaged, e.g., in vivo in an animal or human by introducing into the cells a nucleic acid encoding an internalizing receptor, administering to the animal or human a reporter complex including one or more receptor-specific reporter moieties linked to one or more reporter groups, such as magnetic particles, and detecting the reporter complex, e.g., using magnetic resonance imaging, and thus detecting the cells. If a specific gene is expressed in a constant, known ratio compared to expression of the receptor, e.g., if expression of the two genes is linked, the expression of that gene can be monitored by detecting the reporter complex, and thus, concomitantly, expression of the internalizing receptor and the specific gene.
Accordingly, the invention generally features a methods of imaging expression of a specific gene, e.g., a therapeutic gene such as one that encodes an enzyme, in vivo in a subject, by introducing a nucleic acid encoding an internalizing receptor and the specific gene into cells in the subject; administering to the subject a reporter complex comprising a receptor-specific moiety and a reporter group, wherein the reporter complex binds to the internalizing reporter; and imaging the subject to monitor the reporter complex as an indication of gene expression. For example, the nucleic acid encoding the internalizing receptor can be in a viral or nonviral vector. The imaging can be, for example, magnetic resonance imaging, NMR spectroscopy, or nuclear imaging.
In a specific example, the internalizing receptor is a transferrin receptor, and the reporter complex comprises transferrin and one or more magnetic, paramagnetic, or superparamagnetic nanoparticles. In another example, reporter group is a magnetic particle, an optically detectable molecule, or a radioisotope. In addition, the internalizing receptor can be genetically modified, e.g., to alter recycling of the receptor, internalization, ligand affinity, or receptor half-life within the cell.
In certain embodiments, the reporter complex includes one or more cross-linked iron oxide nanoparticles (CLIOs) or monocrystalline iron oxide nanoparticles (MIONs).
In another aspect, the invention features nucleic acid constructs including a nucleic acid sequence encoding an internalizing receptor, e.g., a transferrin receptor; and a specific gene, such as a therapeutic gene, for example one that encodes an enzyme that metabolizes a drug. The construct can be non-down-regulatable. The gene can also encode a gene product, e.g., for replacement gene therapy, such as the p53 gene. The construct can further include one or more regulatory sequences. For example, the regulatory sequence can include a promoter (e.g., a bicistronic construct) or two promoters, that can be the same or different. In certain embodiments, the promoter induces expression without regulation by environmental conditions within a cell. In certain embodiments, the nucleic acid encoding the receptor can be genetically modified.
The invention also features a viral or nonviral vector, or other nucleic acid delivery vehicle, that includes the new nucleic acid constructs.
In another aspect, the invention also features a reporter complex including one or more internalizing receptor-specific moieties and one or more reporter groups, e.g., magnetic, paramagnetic, or superparamagnetic particles, e.g., monocrystalline iron nanoparticles (MIONs) or cross-linked dextran coated iron oxide nanoparticle (CLIOs), or optically detectable molecules (e.g., fluorescent molecules (e.g., FITC or rhodamine), near infrared molecules (e.g., Cy5), or autoquenching molecules), or radioisotopes (e.g., iodine-125, Tc-99, In-111, or Fe-59). The complex can also include a linker molecule that connects the receptor-specific moiety to the reporter group. The linker is bi- or multi-functional and thus has at least two reactive groups that bind to the reporter group and the receptor-specific moiety. The linker can provide a spacer between the linked reporter group and the moiety.
In another aspect, the invention features a method of inducing cells to internalize magnetic particles, by introducing into the cells a nucleic acid encoding an internalizing receptor; and contacting the cells with a reporter complex comprising a receptor-specific moiety and a reporter group, whereby the moiety binds to the receptor and the complex is moved into the cell carrying the reporter group. The reporter group can be a magnetic particle, an optically detectable molecule, or a radioisoptope.
The invention also features transgenic animals and cell lines that include the new nucleic acid constructs. In addition, the invention includes imaging kits that contain the new nucleic acid constructs and reporter complexes.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The invention provides numerous advantages. For example, the invention provides real time imaging of gene expression in vivo at high spatial resolutions, which enables the study of both endogenous and exogenous (e.g., gene therapy, such as therapeutic, e.g., short-term, gene therapy or long-term, replacement gene therapy) gene expression in live animals and in human clinical studies. In addition, the new methods are noninvasive, and enable repeated longitudinal studies within same animal or human patient.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.